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Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

ABSTRACT: During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC.

SUBMITTER: Caraballo JC

PROVIDER: S-EPMC3075095 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

Caraballo Juan Carlos JC Yshii Cecilia C Butti Maria L ML Westphal Whitney W Borcherding Jennifer A JA Allamargot Chantal C Comellas Alejandro P AP

American journal of physiology. Lung cellular and molecular physiology 20110121 4

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithel ...[more]

PMID: 21257729

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Project description:Protein kinase C (PKC), a widely-distributed enzyme implicated in the regulation of many physiological processes, consists of a family of at least twelve isoenzymes which differ in tissue distribution, subcellular localization, regulatory properties, etc. In addition to this heterogeneity at the protein level, we identify here for the first time a PKC zeta pseudogene (psi PKC zeta) transcript, specifically expressed in the brain, which is identical with PKC zeta except for sequence divergence within the first variable domain (V1). The authenticity of this unique V1 sequence (V1') in mRNA was confirmed by RNase protection and reverse transcriptase PCR (RT-PCR) analysis. When translated in-frame with PKC zeta, a stop codon is located 28 amino acids towards the N-terminus of the divergence point and the intervening sequence lacks an expected initiating methionine. psi PKC zeta is non-functional in terms of protein synthesis since Western blotting with an antibody directed against the C-terminus of PKC zeta failed to reveal a protein smaller than PKC zeta, and synthetic psi PKC zeta RNA failed to support protein synthesis in a translation system in vitro. PCR amplification of rat genomic DNA demonstrated lack of an intron at the junction between V1' and the first constant domain (the V1'-C1 border), and genomic DNA Southern blot analysis using PKC zeta and psi PKC zeta-specific probes indicated that they have different loci. psi PKC zeta, therefore, is not derived from the PKC zeta gene by alternative splicing, but rather is the product of a distinct gene. In Northern blot analysis, brain PKC zeta mRNA was identified as a low-abundance 3.1 kb transcript, while the abundant 2.5 and 4.7 kb mRNAs previously reported to encode PKC zeta are, in fact, psi PKC zeta transcripts. Analysis of rat brain, heart, lung, liver, kidney and skeletal muscle revealed psi PKC zeta mRNA only in brain. PKC zeta transcripts were most abundant in lung and kidney (2.7 and 4.7 kb mRNAs), correlating with the tissue profile of PKC zeta immunoreactivity in Western blots. Probes complementary to the common V5 and C1 domains detected both PKC zeta and psi PKC zeta transcripts. Interestingly, the C1 probe also detected an abundant novel 1.75 kb mRNA in brain and heart, suggesting the existence of an additional PKC zeta-related species. This work, therefore, also emphasizes the importance of careful choice of oligonucleotide and cDNA probes to study PKC zeta mRNA.

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Dataset Information

Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

Publications

Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

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